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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:641-649 (2006)
© 2006 The Gerontological Society of America

T-Kininogen: A Biomarker of Aging in Fisher 344 Rats With Possible Implications for the Immune Response

Claudio Acuña-Castillo, Elias Leiva-Salcedo, Christian R. Gómez, Viviana Pérez, Min Li, Claudio Torres, Robin Walter, Donna M. Murasko and Felipe Sierra

1 Instituto de Ciencias Biomédicas, Programa de Biología Celular y Molecular, Facultad de Medicina, Universidad de Chile, Santiago.
2 Centro FONDAP de estudios moleculares de la célula, Facultad de Medicina, Universidad de Chile, Santiago.
3 Lankenau Institute for Medical Research, Wynnewood, Pennsylvania.
4 Department of Bioscience and Biotechnology, Drexel University, Philadelphia, Pennsylvania.

Address correspondence to Felipe Sierra, PhD, National Institute on Aging, 7201 Wisconsin Ave., Suite 2C231, Bethesda, MD 20892. E-mail: sierraf{at}nia.nih.gov


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
T-kininogen (T-KG) is a reliable biomarker of aging in male Sprague-Dawley rats. Here we confirm, in a longitudinal study, a similar behavior in Fisher 344 rats of both sexes. In males, the increase in serum levels of T-KG follows an exponential curve, whereas in females the increase is best fitted by a linear curve. In both genders, dietary restriction delays the increase in T-KG. We have previously shown that T-KG inhibits T lymphocyte proliferation. Here we show that serum T-KG levels correlate negatively with the ability of splenocytes (most likely B cells) to proliferate in response to lipopolysaccharide. A similar correlation was not observed with other markers of inflammation, including {alpha}1-acid glycoprotein (AGP), haptoglobin, or interleukin-10. We conclude that the increase in serum T-KG represents a useful biomarker of aging in Fisher 344, and it correlates with decreased lymphocyte proliferation with age, although a cause–effect relationship has not been established.

THE aging process is complex in nature, and its study at the molecular level is further confounded by the existence of age-associated diseases. Animal models of this process have been used with varied success over the years (1–6). Like their human counterparts, however, individual animals age at widely different rates; therefore, they will manifest the symptoms and characteristics of the aging process in a rather idiosyncratic fashion (7,8). For this reason, chronological age is not necessarily a good indicator of physiological age. These considerations support the need to develop adequate physiological biomarkers of the aging process in the animal models most currently used in the field.

In the past, we have reported on the identification of T-kininogen (T-KG) as a gene whose hepatic expression is strongly induced during aging in male Sprague–Dawley rats (9,10). A cross-sectional study in which hepatic levels of T-KG messenger RNA (mRNA) were measured in Fisher 344 (F344) and (F344 x Brown Norway)F1 rats of both sexes indicated that T-KG appears to be a promising candidate molecular biomarker of aging in these animals as well (11). T-KG mRNA levels increase between 5-fold and 10-fold as a function of age, and this increase is delayed under caloric restriction, an experimental intervention known to delay aging (11). T-KG is a secreted molecule, and although the age-related increase in serum T-KG levels is less pronounced than the increase in hepatic mRNA, it is clear that measuring serum proteins has the advantage of being less invasive than measuring hepatic mRNA. For this reason, we have undertaken to measure the increase in serum levels of T-KG in a longitudinal study in F344 rats. We have used both male and female rats, and in both cases animals were separated into two cohorts either receiving food ad libitum (AL) or under a dietary restriction regime (DR).

The usefulness of biomarkers is greatly increased when they can be shown to be an integral part of the mechanism underlying the process in question. Thus, for example, hair graying is a good biomarker of aging in humans, but the recent demonstration that hair graying is the result of preferential apoptosis of the melanocyte stem cell precursors (12) makes this biomarker considerably more useful for studies of age-related mechanisms. In the case of T-KG, we have shown that its overexpression in fibroblasts leads to an impairment in extracellular regulated kinase (ERK) activation (13) and cell proliferation (14). In contrast, when applied exogenously, the effect of T-KG is highly cell-specific, being inhibitory of both ERK and proliferation in the case of T cells (15), but stimulatory of both of these activities when added to either endothelial cells (16) or fibroblasts (17).

There is considerable evidence in the literature suggesting that the immune system deteriorates with age, rendering the aged less able to mount an immune response (18). Whereas changes have been reported in both the innate and adaptive parts of the response, most of the deterioration described to date occurs in the adaptive response of T and B lymphocytes (19). Specifically, molecular and cellular defects, which lead to a reduced proliferative capacity, have been described in studies of T lymphocytes from aged humans as well as from animal models (20). Mechanistically, impairments have been described at several levels of the signal transduction machinery, including several kinases associated directly with the CD3 complex, as well as kinases downstream, such as mitogen-activated protein kinases (MAPKs) [reviewed in (21)]. T-KG can negatively modulate the responsiveness of T cells to nonspecific stimulation (15). Here, we chose to test whether the level of T-KG in vivo correlates with the ability of splenocytes to proliferate in response to an in vivo injection of lipopolysaccharide (LPS). LPS is a powerful mitogen for many cells of the immune system, including T cells (22,23). However, the best described proliferative effect occurs in cells of the B lineage (24). Our results indicate a strong negative correlation between the levels of serum T-KG and the proliferative capacity of splenocytes isolated from the same animals. This correlation is stronger than that observed for other markers of inflammation, such as {alpha}1-acid glycoprotein (AGP), haptoglobin, or interleukin-10 (IL-10). We conclude that T-KG is a bona fide biomarker of aging in rats, and its levels correlate negatively with splenocyte proliferation.


   METHODS
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 Methods
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 Discussion
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Animals
Both male and female F344 rats were obtained from the National Institute on Aging, National Institutes of Health (NIH). All animals were housed individually in microisolator cages within our barrier facility. Rats were given NIH 31 pellets, either AL, or DR to 60% of the average intake of AL rats. The restricted diet was NIH 31 pellets supplemented with vitamins and minerals. Restriction began at weaning and was gradually reduced until the level stabilized at 60% by 4 months of age. Mean life span for these animals is 24 and 29 months for males (AL and DR, respectively), and 27 and 31 months for females (AL and DR, respectively).

Sample Preparation
For the longitudinal studies and analysis of complexes containing T-KG, blood was collected retroorbitally in the absence of anticoagulants every 2–3 months, starting at approximately 10–11 months of age. Previous studies have shown that T-KG levels do not start increasing until well after this age; therefore, we considered the level observed at this earliest point as "basal level" (10). As the end of the expected lifetime approached, samples were collected more often (once a month). Serum was prepared by centrifugation at 2000 g for 5 minutes, 1 hour after blood collection, and immediately frozen at –20°C until use. After the natural death of each individual animal, all its serum samples were diluted 1:1000 in phosphate-buffered saline, and 10 µl of the resulting solution was loaded into a single 7.5% mini sodium dodecyl sulfate–polyacrylamide gel for electrophoresis. After electrophoresis and electro transfer to nitrocellulose membranes, the blots were probed with antirat T-KG antibodies (a gift from Dr. Jack Gauldie, University of Ontario, Canada).

Analysis of Complexes Containing T-KG
We used sera from seven independent male F344 rats fed ad libitum, aged either 16 or 22 months. For this analysis, 100 µl of serum was applied to Microcon C-100 columns (Amicon, Beverly, MA) and centrifuged at 6000 g for 15 minutes at room temperature. The filtered fractions were collected for western blotting, and the retained fractions (>100 kd) were applied to a new Microcon C-100 column, centrifuged as before, and the retentates were collected for western blotting. The samples were then diluted 1:500, and 10 µl was analyzed by standard 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and was immunoblotted with anti-T-KG antibodies. As controls, pools of sera were treated as before or were diluted 1:100 before Microcon C-100 separation.

Inflammation and Lymphocyte Proliferation Assays
Rats of different ages (6, 15, and 23 months old) were injected i.p. with LPS at 2 mg/kg and were killed at different times by decapitation, and blood and spleens were collected. The blood was allowed to clot, and aliquots of the serum were stored until use. Proper dilutions of sera were analyzed by western blot as described, using antibodies against T-KG, AGP (also donated by Dr. Jack Gauldie), and haptoglobin (Sigma, St. Louis, MO). Detection was done by using the enhanced chemiluminescence (ECL) system of Boehringer (Ingelheim, Germany). Detection and autoradiography were performed exactly as suggested by the vendor. Quantitation was performed using ImageQuaNT software (Amersham, Sunnyvale, CA). IL-10 was measured in the sera by using a commercial enzyme-linked immunosorbent assay kit, using the conditions suggested by the manufacturer (BD Pharmingen, San Jose, CA).

From the same animals, spleens were removed under sterile conditions, and total splenocytes were isolated as previously described (25). After plating in RPMI-1460 medium, cells were diluted and seeded at 1 x 105 cells/well in 96-well plates. After 24 hours, 200 nCi of [3H]-TdR were added for 4 hours, cells were pelleted and lysed with 10% trichloroacetic acid (TCA), and [3H]-TdR incorporation was evaluated in a scintillation counter.

Statistical Analysis
For the longitudinal study, all comparisons were performed exclusively within a single animal, and we assigned a value of 1 to the level of serum T-KG observed in each individual animal at the time the first serum sample was obtained. Serum levels of T-KG at all other ages were obtained relative to that value. After grouping by either age or fraction of life span, statistical significance was assessed by Student's t test. For serum levels of acute phase proteins, all samples were normalized against an internal control containing equal amounts of sera obtained at 0 and 12 hours after LPS, from a pool of all animals. For the inflammation portion of the study, statistical significance of measurements performed on individual animals was assessed using the test of Friedman and Quade. For comparisons between treatments, or to establish the effect of age on complex formation, we used the Mann–Whitney U test (Prism 3.0 package; Graphpad, San Diego, CA). Correlations were evaluated with a linear regression analysis, and slope significance was evaluated with a Fisher null hypothesis test, which considers the probability that the slope is different from zero.


   RESULTS
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 Abstract
 Methods
 Results
Discussion
 References
 
Patterns of Age-Dependent Increase in Serum T-KG Levels in F344 Rats
We analyzed a cohort of 32 male and 25 female rats, divided approximately equally between AL and DR (17 male and 12 female rats were under DR). Serum samples were collected starting at age 10–11 months. Collection was initially every 3 months, and towards the time when an inflection in the survival curve was expected, this frequency was increased to once a month. Care was taken to analyze all samples from each individual animal in a single experiment. As an example, Figure 1 shows the results obtained in representative animals. In 67% of all the animals (both sexes and diets combined), serum T-KG levels showed an exponential increase (as shown in A). In 33% of the animals analyzed, however, the increase was more linear (as represented in B). The distinction between these two patterns was made for each individual animal by comparing the r2 values for each kind of curve. It is interesting that the pattern of serum T-KG increase showed a slight sex dependence, with most male animals (both AL and DR) showing an exponential increase, whereas in females this increase was more often linear. Three animals in the whole cohort showed an anomalously high point earlier in life, within a pattern of exponential increase with age. The anomalous point was assumed to be due to a brief acute inflammation; in these three cases, the anomalous point was eliminated, but the rest of the data for the animal were retained for further analysis.


Figure 01
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  		Figure 1. Representative results of the longitudinal analysis. Representative gels (left) and their corresponding quantitations (right) are shown. A, Animal that displays an exponential increase in T-kininogen levels towards the end of life span. B, Animal with a linear increase throughout the time period analyzed. In both cases, animals were diet-restricted males, and these data are shown solely as examples of the types of kinetics observed

 
Serum T-KG Levels Increase With Age in Male F344 Rats, and This Increase Is Delayed by DR
Data on serum levels of T-KG obtained from male F344 rats fed either AL or DR are presented in Figure 2. Animals were grouped either according to chronological age (A) or according to the fraction of life span completed at the time of analysis (B). The data indicated an exponential increase in serum T-KG levels in animals of both groups (r2 = 0.863 and 0.959 for exponential regression curves on AL and DR data, p =.005 and 6 x 10–5, respectively). The data were most significant when the oldest age considered was at 90% of life span, as we often observed a slight decrease in serum T-KG when animals close to their death were analyzed (Table 1 and data not shown). The magnitude of the increase is approximately 3-fold to 4-fold in both groups of animals. DR significantly delays, but does not abolish the age-related increase in serum T-KG levels. When analyzed as a fraction of life span completed, the same general trend is maintained, although the difference between the AL and DR animals is less striking. Statistical analysis of these results is presented in Table 1, which shows that when analyzed as a fraction of life span, the increase in T-KG levels becomes statistically significant at the time when animals have reached either 80% or 60% of their life span for AL and DR rats, respectively. This finding suggests that DR has a composite effect, as it results in both a delay and a reduction (but not elimination) of the increase in serum T-KG levels in male F344 rats.


Figure 02
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  		Figure 2. Serum T-kininogen (T-KG) levels increase with age in male Fisher 344 (F344) rats. Results obtained after measuring serum levels of T-KG in individual male F344 rats were pooled according to either chronological age (A) or as a fraction of life span completed (B). Closed circles: ad libitum fed (AL); open circles: dietary restricted (DR). All data are expressed as relative levels, where a value of 1 was assigned to the level of T-KG found in the earliest sample analyzed (10 or 11 months). Data are presented as average ± standard error of the mean, and the statistical analysis by Student's t test is presented in Table 1

 

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  		Table 1. Statistical Analysis of Male Rat T-Kininogen Levels.

 
Serum T-KG Levels Increase With Age in Female F344 Rats, and This Increase Is Not Significantly Affected by DR
A similar analysis has been performed on female F344 rats fed AL or DR. Figure 3 and Table 2 indicate that in female rats, as in male rats, there is a significant increase in serum T-KG levels as a function of age. For female rats, the best fit was by a linear regression curve (r2 = 0.9515 and 0.8883 for AL and DR animals, p =.011 and.003, respectively), as opposed to the exponential increase observed in male rats. Also, the increase in female animals is less pronounced (approximately 2-fold) than that observed in male animals. This is most likely due to the fact that hepatic levels of T-KG mRNA are 2-fold to 3-fold higher in young female AL rats, as compared to male counterparts (11). As in male rats, DR appears to delay slightly the increase in serum T-KG levels. Again, this effect is less pronounced in female rats, probably due to the aforementioned increase in T-KG gene expression in young female rats (26,27). Analysis of the results as a function of fraction of life span completed indicates that, in female rats, DR has only a borderline effect on the appearance of the marker, and it does not affect the magnitude of the increase.


Figure 03
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  		Figure 3. Serum T-kininogen (T-KG) levels increase with age in female Fisher 344 (F344) rats. The results obtained after measuring serum levels of T-KG in individual female F344 rats were pooled according to either chronological age (A) or as a fraction of life span completed (B). Closed circles: ad libitum fed (AL); open circles: dietary restricted (DR). All data are expressed as relative levels, where a value of 1 was assigned to the level of T-KG found in the earliest sample analyzed (10 or 11 months). Data are presented as mean ± standard error of the mean, and the statistical analysis by Student's t test is presented in Table 2

 

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  		Table 2. Statistical Analysis of Female Rat T-Kininogen Levels.

 
Serum Levels of Free T-KG Increase With Aging
The closely related high-molecular-weight (HMW) kininogen is found primarily in complexes with other serum proteins, and its biological activity depends on its state of association into such complexes (28). Therefore, we measured whether the T-KG found in rat serum is free or in complexes. Figure 4A shows that, with age, there is a significant increase in both the bound and free forms of T-KG. Notably, the free form increased more than the bound, suggesting that the serum of old rats contains a higher level of biologically active kininogens.


Figure 04
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  		Figure 4. A, Serum levels of free T-kininogen (T-KG) increase with age in male Fisher 344 (F344) rats. Relative amount of T-KG present either free or in complexes larger than 100 kd. Top: Quantitation from 7 individual rats of each age, either 16 (white bars) or 22 (gray bars) months old. Data are presented as average ± standard error of the mean, and statistical significance was assessed by the unpaired Mann–Whitney U test. Bottom left: representative western blot, showing both free (F) and Complexed (C) T-KG, either in 16-month-old or 22-month-old rats. Bottom right: control in which all noncovalent complexes were disrupted by dilution (1:500) in phosphate-buffered saline prior to separation in the Centricon C-100 column. All the T-KG appears as free. B, Response of serum T-KG levels to lipopolysaccharide (LPS) in male F344 rats. Rats of different ages (6 months, triangles; 15 months, gray circles; 23 months, black circles) received injections of LPS as described and were killed at the time indicated, and serum T-KG was measured by western blot as before. Inset: induction of T-KG 24 hours after LPS challenge in 6-month-old (white bar), 15-month-old (gray bar), and 23-month-old (black bar) rats. All data are presented relative to an internal standard consisting of a pool of samples from several animals. Top: Quantitation from five individual rats per age group. Bottom: Representative western blot corresponding to one individual rat per time and age point

 
Also relevant in this context is the fact that T-KG is an acute phase reactant, and its circulating levels increase after an inflammatory stimulus. The age-related increase described here and elsewhere is not, however, due to inflammatory processes, as other acute phase proteins do not increase with age (29). However, the inducibility of this protein after an inflammatory challenge has not been investigated in aged rats. Figure 4B indicates that, while basal T-KG is increased in old rats as described, its levels are not further induced within 12 hours after an LPS injection. Induction was indeed observed at 24 hours after LPS challenge, and at this point no difference was observed among animals of different ages (Figure 4B, inset).

T-KG Levels Show a Negative Correlation With Splenocyte Proliferation
We have previously demonstrated that purified T-KG inhibits the proliferation of T lymphocytes (15). T lymphocyte proliferation is often reduced in old rodents, but little is known about the effect of age on the proliferative capacity of other cell types within the immune system. LPS is a nonspecific inducer of proliferation in a variety of cells, and for that reason we chose LPS as a general inducer. Animals of different ages received LPS injections in vivo, total splenocytes were isolated at different times and cultured for 20 hours, and then [3H]-thymidine was added for 4 additional hours. Figure 5A shows that the systemic challenge with LPS led to a proliferative response of splenocytes, but mainly in the 6-month-old and 15-month-old groups. A significantly blunted response was observed in the aged rat group. In Figure 5B, these same data were plotted against the serum level of different markers of inflammation, as measured at the time of death. The data indicate a negative correlation between serum T-KG levels and thymidine incorporation (slope = –784 ± 280, r2 = 0.193, p =.007). In contrast, we did not observe a significant correlation when other acute phase proteins were analyzed. Although haptoglobin levels also show a negative slope (–159 ± 102), this effect was not statistically significant (r2 = 0.067, p =.127), and serum AGP levels did not show any correlation with thymidine incorporation (slope = –34 ± 274, r2 = 0.0003, p =.901). We also measured serum levels of the immunosuppressor cytokine IL-10. As shown in Figure 5B, IL-10 did not show a correlation with thymidine incorporation either (slope = 0.24 ± 0.37, r2 = 0.01, p =.517). Thus, we conclude that, of the parameters measured, only T-KG levels correlate significantly with decreased splenocyte proliferation.


Figure 05
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  		Figure 5. Serum T-kininogen (T-KG) levels correlate with a diminished proliferative response in splenocytes from rats exposed to lipopolysaccharide (LPS). Male rats of different ages (6 months, triangles; 15 months, white circles; 23 months, black circles) received injections of LPS as described and were killed between 0 and 12 hours postchallenge. A, Splenocyte proliferation was analyzed without any further stimulus, and [3H]-TdR incorporation was measured for a period of 4 hours, 24 hours after isolation. B, Blood was collected from the same animals, and serum was analyzed for T-KG, {alpha}1-acid glycoprotein (AGP), and haptoglobin by western blot and for interleukin-10 (IL-10) by enzyme-linked immunosorbent assay, as described in Methods. Proliferation values were correlated animal-by-animal with the values obtained for the inflammatory markers. We used at least 12 animals per age group, but age was not included as a variable in this analysis, so the data presented are combined for all the animals and times after LPS treatment. Curves were fitted with a linear equation, and correlation analysis was evaluated using the r2 value. Significance was assessed by analyzing the probability that the slope is different from 0, using a Fisher test

 

   DISCUSSION
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References
 
Molecular biomarkers of the aging process have long been sought by the research community, with little success. One of the earliest molecular biomarkers described was {alpha}2 macroglobulin, a liver protein whose expression in rats decreases dramatically in aged rats and mice, primarily as a consequence of its dependence on androgens (30). Also, based primarily in T-cell subsets, Miller and his group (31,32) have identified some immune markers as validated predictors of longevity. Furthermore, using four-way crosses of mice, the same group has further defined several additional such predictors of longevity (33). Recent developments on the genetics of aging show the promise of unveiling yet other biomarkers, such as members of the insulin-like growth factor/insulin axis (34). However, no definitive studies in this direction have been published thus far. Although it is unlikely that any single protein or metabolite will by itself represent a perfect biomarker of aging, the data presented here support the notion that serum levels of T-KG represent a reasonably good candidate biomarker, at least in F344 rats. It should be kept in mind, however, that age-related pathology could complicate our interpretation. We did not perform thorough autopsies in any of the animals used in this study. However, the fact that serum T-KG levels (but not other acute phase reactants; 29), increase with age in almost all individuals from a variety of rat strains that succumb to different diseases, allows us to be confident that this is an age-related phenomenon, rather than a disease-related one. For each individual animal, the data have been standardized to the level observed at the youngest age tested (10–11 months), and thus represents a fold increase, rather than absolute levels of T-KG in the serum. Expressed in this manner, serum T-KG levels increase quite dramatically, particularly in male animals, and this increase is delayed, but not abolished by DR, as expected. In female animals, the increase is less pronounced, probably due to the confounding upregulation of its expression by estrogens (26,27). However, this is only a partial explanation, as the age-related increase still attains only 2-fold in DR female animals, whose estrogen levels are depressed compared to those of AL female animals, and their basal levels of hepatic T-KG mRNA are not significantly different from those observed in males of a similar age and diet (11). It is likely, therefore, that further hormonal influences, possibly affecting protein stability and turnover, may also play a role in the attenuated response of the parameter in female rats. Although this and other studies from our group (9,11) suggest that T-KG is a reasonable biomarker of aging in several strains of rats, it should be mentioned that the gene exists solely in this species. However, it has been shown that serum levels of another member of the gene family, namely HMW-KG, increase as a function of age both in rats (5) and in humans (35).

T-KG is produced primarily in the liver, and the age-related increase in serum levels is driven largely by an increase in transcription of the gene in this tissue (9). T-KG accumulates in hepatocytes from old rats, but a substantial amount is also secreted into the bloodstream, giving rise to the increase described in this study. The physiological role, if any, of the age-related increase in serum T-KG is not currently understood. We have previously shown that T-KG can affect the physiology of several blood-borne cells. Specifically, exposure of endothelial cells (16) or fibroblasts (17) to exogenous T-KG leads to a robust proliferative response, driven at least in part by activation of the ERK pathway. In contrast, exposure of lymphocytes to exogenous T-KG leads to inhibition of both basal and Concanavalin A-induced proliferation (15). Most studies indicate that T lymphocytes derived from old rodents or humans display a reduced proliferative response to mitogens (reviewed in 18). Because T-KG can inhibit this response, at least in vitro (15), in this study we addressed the possibility that the proliferative response of total splenocytes in response to a nonspecific systemic challenge (LPS) might be correlated with serum levels of T-KG. We first investigated whether the levels of free T-KG (the purported biologically active entity) increase with age. Whereas total T-KG levels increase, free T-KG could at least theoretically remain unchanged if its binding partners were in excess, and complex formation did not follow simple reaction kinetics. However, Figure 4A indicates that, in fact, it is the free form of T-KG that increases the most as a function of age. This finding suggests that, in the complexes formed, T-KG might be rate-limiting. In the case of HMW-KG, it circulates as a complex together with kallikrein and Hagemann factor (28). However, the proteins that form complexes with either low-molecular-weight (LMW) KG or T-KG are not currently known. Whereas LMW KG is also a substrate for kallikrein, T-KG is not, and thus it is unlikely that this particular protease will form part of the complexes we have detected by size filtration. Having established that free T-KG levels increase in the serum of old rats, we wanted to test whether this increase correlated with the ability of splenocytes to proliferate in response to LPS. Figure 5 shows that splenocytes proliferate in vitro in response to an in vivo injection of LPS, and that this response is blunted in old rats. For these experiments we used total splenocytes, and it has been shown that several lymphocyte classes, including both B and T lymphocytes, as well as dendritic cells, are capable of proliferation in response to LPS (22–24). LPS induces a complex immune response which also includes expression of acute phase proteins, including T-KG. Within the time of the experiment, however, serum T-KG levels increased in only young and middle-aged rats, not in old rats. Perhaps more importantly, when age was not considered as a variable, and the proliferative response was correlated with the serum levels of a variety of inflammatory markers, we found that the reduced proliferation only correlates significantly with T-KG levels, but not with levels of AGP, haptoglobin, or IL-10. Although these studies are solely correlative, and do not show cause and effect, they are consistent with the results obtained in vitro, with lymphocytes exposed directly to free T-KG (15). These correlations must be considered in conjunction with other data showing the effect of age on the acute phase response. In rats, we have reported no age-related changes in the steady-state mRNA levels of AGP or {alpha}2-macroglobulin (29). In contrast, using mice, the group of Papaconstantinou has shown a significant decrease with age in the constitutive levels of {alpha}1-antitrypsin mRNA, as well as an age-dependent decline (and delay) in the ability of the AGP and albumin genes to respond to inflammation (36,37). Furthermore, the same group has shown changes in the ability of mouse cardiac tissue to induce several cytokines in response to systemic LPS. For example, although no age-associated differences were observed on the induction of tumor necrosis factor-{alpha} mRNA, the induction of IL-1ß was modestly extended, and the induction of IL-6 was significantly prolonged in aged mice, compared to younger controls (38).

We do not presently know the mechanism by which T-KG inhibits the proliferation of splenocytes. T-KG is a precursor of kinins, and is a potent cystatin-like cysteine proteinase inhibitor. The age-related increase in serum T-KG correlates with increased levels of free kinins (39), and this increase might play a role in the increased algesia and vascular disorders often observed in aging individuals. However, kinins have been shown not to have an effect on lymphocyte proliferation, at least within physiological ranges (40,41), and at least in vitro, the inhibitory effect of T-KG on T-lymphocyte proliferation (15) does not require kinin receptors (Acuña-Castillo C, Sierra F, unpublished observations, 2002). In contrast, cystatins have been described as inhibitors of T-cell proliferation, capable of inducing a switch from a type 1 (Th1) to a type 2 (Th2) response (reviewed in 42), similar to what has been described to occur during aging (reviewed in 43). Specifically, this switch appears to be the mechanism used by several filarial worms, which secrete potent cystatins to neutralize the immune response of the host [reviewed in (42,44,45)]. It is very likely that T-KG inhibits the immune response by a mechanism comparable to the one used by filarial worms. Unfortunately, very little is known about this mechanism at the molecular level.

Summary
Our results indicate that serum levels of T-KG can serve as a valuable biomarker of aging in F344 rats. Furthermore, although the current data are admittedly only correlative, they suggest that the increase in this molecule could also have a dramatic physiological effect, possibly playing a role in the well-known depression in proliferative capacity of lymphocytes in response to inflammatory stimuli.


   Acknowledgments
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This research was funded by FONDECYT (grants 2010071, 2000038, and 1010615), MECESUP (RUch 9903), FONDAP 15010006, and the National Institutes of Health/National Institute on Aging (R01 AG 13902 and R01 AG 7719).


   Footnotes
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Decision Editor: James R. Smith, PhD

Received July 19, 2005

Accepted December 14, 2005


   References
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